How insulin binds to its receptor defines a central problem in molecular endocrinology. This competing application seeks to define the active structure of insulin and points of contact between the hormone and the a subunit of the insulin receptor (IR). We will next exploit the recent crystal structure of the receptor ectodomain to test whether insulin binding triggers reorganization of the ectodomain's novel inverted-V conformation. As a bridge between structure and function, in vitro evolution of the insulin receptor will be undertaken to obtain altered ligand specificity. We envisage that altered-specificity hormone-receptor pairs will enable a novel strategy to investigate tissue-specific insulin signaling in transgenic mice. Aim 1 focuses on non-standard structure-activity relationships in insulin through 'chiral mutagenesis': comparison of corresponding D- and L-amino-acid substitutions at proposed sites of conformational change. This strategy exploits chemical synthesis of insulin to test the hypothesis that the B-chain reorganizes on receptor binding. This model will be tested through time-resolved FRET studies of insulin derivatives containing a fluorescent donor and acceptor bridging proposed sites of conformational change. Aim 2 seeks to define points of hormone-receptor contact by two approaches: (a) site-specific photo-cross- linking based on para-azido-Phe insulin derivatives;and (b) restoration of binding between otherwise inactive insulin analogs by alanine scanning mutations in the receptor a subunit. Mapping of photo-products will be accomplished using ectodomain constructs designed by D. F. Steiner (Univ. of Chicago) and tandem-MS in the Case Center for Proteomics &Mass Spectrometry. By determining multiple points of hormone-receptor contact, a molecular model of the insulin-ectodomain complex will be constructed. Aim 3 investigates whether insulin binding triggers a conformational change in the IR ectodomain. Experimental design builds on the recent inverted-V crystal structure of the free ectodomain. Through novel protein engineering strategies, we will test whether the spatial relationship between the splayed legs of the ectodomain is altered on binding of insulin. In these studies the DMA double helix will be employed as a "molecular ruler" to measure leg spacing in an optimal inverted-V ectodomain conformation. Aim 4 seeks to define altered-specificity pairs of hormones and receptors. The essential idea is to employ an inactive insulin analog to evolve a receptor variant that binds and responds only to that analog and not wild-type insulin. Chemical synthesis of fluorescently labeled mutant insulins will enable screening for compensating receptor mutations in a FACS-based assay. Random-cassette mutagenesis of the a subunit will be guided by the results of Aim 2. Aim 4 promises not only to illuminate principles of receptor specificity, but also to enable novel physiological studies in transgenic mice. To this end, "bait" insulin analogs will be chosen to have otherwise native structures, stabilities, and assembly properties - therefore to be appropriate for pharmaceutical administration to mice. As a long-term objective, we envisage introduction of a "private-label" insulin signaling system in the background of a Kahn tissue-specific IR knock-out mouse. To demonstrate proof-of- principle, respective application of this enabling technology to the liver and pancreatic p cells of LIRKO and PIRKO mice is planned in collaboration with C. R. Kahn (Joslin Diabetes Center, Boston).